3-(10′-Phenothiazinyl)propane-1-sulfonate is a potent enhancer of soybean peroxidase-induced chemiluminescence

3-(10′-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of soybean peroxidase (SbP)-induced chemiluminescence. To the best of our knowledge, SPTZ is the first enhancer of SbP to be discovered. Optimal conditions for SbP-catalyzed oxidation of luminol in the presence of SPTZ were determined. The SbP-SPTZ system showed better sensitivity and a lower detection limit (LDL) with respect to the horseradish peroxidase-4-iodophenol system traditionally used in chemiluminescent enzyme-linked immunosorbent assay (ELISA). The addition of 4-morpholinopyridine (MORP) to the SbP-SPTZ system improved its analytical parameters by decreasing the LDL of SbP to 0.03 pM. These results open up very promising perspectives for using the SbP-SPTZ-MORP system in ultrasensitive immunoassay.     

Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP‐C) in the presence of 3‐(10'‐phenothiazinyl) propane‐1‐sulfonate (SPTZ) and 4‐morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP‐catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N‐azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd.     

Luminol-hydrogen peroxide chemiluminescence produced by sweet potato peroxidase.

Anionic sweet potato peroxidase (SPP; Ipomoea batatas) was shown to efficiently catalyse luminol oxidation by hydrogen peroxide, forming a long-term chemiluminescence (CL) signal. Like other anionic plant peroxidases, SPP is able to catalyse this enzymatic reaction efficiently in the absence of any enhancer. Maximum intensity produced in SPP-catalysed oxidation of luminol was detected at pH 7.8-7.9 to be lower than that characteristic of other peroxidases (8.4-8.6). Varying the concentrations of luminol, hydrogen peroxide and Tris buffer in the reaction medium, we determined favourable conditions for SPP catalysis (100 mmol/L Tris-HCl buffer, pH 7.8, containing 5 mmol/L hydrogen peroxide and 8 mmol/L luminol). The SPP detection limit in luminol oxidation was 1.0 x 10(-14) mol/L. High sensitivity in combination with the long-term CL signal and high stability is indicative of good promise for the application of SPP in CL enzyme immunoassay.     

An investigation on the catalytic mechanism of enhanced chemiluminescence: Immunochemical applications of this reaction

The mechanism of peroxidase-catalysed oxidation of luminol by H₂O₂ was studied. The stopped-flow technique was used to measure the rate constants for the reactions between the oxidized forms of peroxidase with luminol and the following substrates: p-iodophenol, p-bromophenol, p-clorophenol, o-iodophenol, m-iodophenol, luciferin, and 2-iodo-6-hydroxybenzothiazole. The correlation between kinetic parameters and the degree of enhancement was established. The effect of charged synthetic polymers and specific antibodies on the peroxidase activity in the enhanced chemiluminescent reaction. Novel homogenous methods of luminescent immunoassay (LIA) for (1) antibodies to insulin, (2) insulin and (3) antibodies to trinitrophenyl group are proposed on the basis of regulatory facilities of the enhanced chemiluminescent reaction. Based on the enhanced chemiluminescent reaction a peroxidase flow-injection assay was developed and successfuly tested in the flow-injection enzyme immunoassays for human IgG and for thyroxin (T4). The immunoassay proposed has a detection limit of 10^?9M for IgG and 10^?11M for T4, the overall time of the assay being 5–15 min.     

Long-Term Chemiluminescent Signal Is Produced in the Course of Luminol Peroxidation Catalyzed by Peroxidase Isolated from Leaves of African Oil Palm Tree

Optimal conditions were found for the oxidation of luminol by hydrogen peroxide in the presence of peroxidase isolated from leaves of the African oil palm tree Elaeis guineensis (AOPTP). The pH range for maximal chemiluminescence intensity (8.3-8.6) is similar for AOPTP, horseradish, and Arthromyces ramosus peroxidases and slightly different from that for tobacco peroxidase (9.3). Increasing the buffer concentration decreases the chemiluminescence intensity. As in the case of other anionic peroxidases, the catalytic efficiency of AOPTP does not depend on the presence of enhancers (4-iodophenol and 4-hydroxycinnamic acid) in the reaction medium. The detectable limit of AOPTP assayed by luminol peroxidation is 2·10^–12 M. The long-term chemiluminescence signal produced during AOPTP-dependent luminol peroxidation is a characteristic feature of the African oil palm enzyme. This feature in combination with its very high stability suggests that AOPTP will be a promising tool in analytical practice.     

Phenol derivatives as enhancers and inhibitors of luminol–H2O2–horseradish peroxidase chemiluminescence

Systematic studies on phenol derivatives facilitates an explanation of the enhancement or inhibition of the luminol–H₂O₂–horseradish peroxidase system chemiluminescence. Factors that govern the enhancement are the one-electron reduction potentials of the phenoxy radicals (PhO^•/PhOH) vs. luminol radicals (L^•/LH⁻) and the reaction rates of the phenol derivatives with the compounds of horseradish peroxidase (HRP-I and HRP-II). Only compounds with radicals with a similar or greater reduction potential than luminol at pH 8.5 (0.8 V) can act as enhancers. Radicals with reduction potentials lower than luminol behave in a different way, because they destroy luminol radicals and inhibit chemiluminescence. The relations between the reduction potential, reaction rates and the Hammett constant of the substituent in a phenol suggest that 4-substituted phenols with Hammett constants (σ) for their substituents similar or greater than 0.20 are enhancers of the luminol–H₂O₂–horseradish peroxidase chemiluminescence. In contrast, those phenols substituted in position 4 for substituents with Hammett constants (σ) lower than 0.20 are inhibitors of chemiluminescence. On the basis of these studies, the structure of possible new enhancers was predicted. © 1998 John Wiley & Sons, Ltd.     

A comparative study of peroxidases from horse radish and Arthromyces ramosus as labels in luminol-mediated chemiluminescent assays.

The properties of a peroxidase from Arthromyces ramosus (ARP) in the chemiluminescent reaction of luminol oxidation have been studied. These were compared with the properties of horse radish peroxidase (HRP) in the cooxidation of luminol and p-iodophenol, the enhanced chemiluminescence (ECL) reaction. By means of the stop-flow technique, ARP was shown to have an enzymatic activity toward luminol higher than that toward HRP. ARP can efficiently catalyze luminol oxidation in the absence of substrate enhancer. pH and substrate concentrations were optimized to determine ARP with the highest sensitivity. The detection limit of ARP was 5 x 10(-13) M, the same as that for HRP in the ECL reaction. The data on the use of ARP as a label in enzyme immunoassay of human IgG are presented. ARP was shown to have all the advantages of HRP as a label in chemiluminescent enzyme immunoassays: (i) high signal intensity, (ii) slow decay of luminescence, (iii) high signal/noise ratio, and (iv) as a consequence of (i)-(iii), high detection sensitivity. However, the low thermostability of ARP can limit the potential fields of its application.     

Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction

The wild-type anionic tobacco peroxidase and its Glu141Phe mutant have been expressed in Escherichia coli, and reactivated to yield active enzymes. A Glu141Phe substitution was made with the tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases, such as horseradish peroxidase (HRP). Both recombinant forms of tobacco peroxidase show extremely high activity in luminol oxidation with hydrogen peroxide, and thus, preserve the unique property of the native tobacco peroxidase, a superior chemiluminescent reagent. The chemiluminescent signal intensity for both recombinant forms of TOP is orders of magnitude higher than that for wild-type recombinant HRP. The substitution slightly increases TOP activity and stability in the reaction course, but has almost no effect on the optimal parameters of the reaction (pH, luminol and hydrogen peroxide concentrations) and calibration plot. Comparison of substrate specificity profiles for recombinant TOP and HRP demonstrates that Glu141 has no principal effect on the enzyme activity. It is not the presence of the negative charge at the haem edge, but the high redox potential of TOP Compounds I and II that provides high activity towards aromatic amines and aminophenols, and luminol in particular.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Study on the reaction mechanism and the static injection chemiluminescence method for detection of acetaminophen

Acetaminophen, also called paracetamol, is found in Tylenol, Excedrin and other products as over–the‐counter medicines. In this study, acetaminophen as a luminol signal enhancer was used in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP) for the first time. The use of acetaminophen in the luminol–HRP–H₂O₂ system affected not only the intensity of the obtained signal, but also its kinetics. It was shown that acetaminophen was to be a potent enhancer of the luminol–HRP–H₂O₂ system. A putative enhancement mechanism for the luminol–H₂O₂–HRP–acetaminophen system is presented. The resonance of the nucleophilic amide group and the benzene ring of acetaminophen structure have a great effect on O‐H bond dissociation energy of the phenol group and therefore on phenoxyl radical stabilization. These radicals act as mediators between HRP and luminol in an electron transfer reaction that generates luminol radicals and subsequently light emission, in which the intensity of CL is enhanced in the presence of acetaminophen. In addition, a simple method was developed to detect acetaminophen by static injection CL based on the enhanced CL system of luminol–H₂O₂–HRP by acetaminophen. Experimental conditions, such as pH and concentrations of substrates, have been examined and optimized. The proposed method exhibited good performance, the linear range was from 0.30 to 7.5 mM, the relative standard deviation was 1.86% (n = 10), limit of detection was 0.16 mM and recovery was 99 ± 4%. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of Enhancers on the Pyridopyridazine–Peroxide–HRP Reaction

4-Substituted phenyl boronic acids (e.g., 4-iodo, 4-bromo, 4-phenyl) are effective enhancers of the horseradish peroxidase (Type VIA) catalysed chemiluminescent oxidation of various pyrido[3,4-d]pyridazine-1,4(2H,3H)dione derivatives. The most effective combination was 4-biphenylboronic acid and 8-amino-5-chloro-7- phenylpyrido[3,4-d]- pyridazine-1,4(2H,3H)dione. Generally, the intensity of light emission in the presence of peroxidase was higher with the pyridopyridazines than with sodium luminol. However, the blank light emission was much lower with sodium luminol than with the pyridopyridazines. A synergistic enhancement phenomenon was demonstrated for the combination of a 4-iodophenol and a 4-biphenylboronic acid enhancer with 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione. The combination of these two enhancers produced a light emission intensity in an assay for 5 fmol of peroxidase that was 25% higher than expected from the sum of the individual light intensities.     

Luminol oxidation by hydrogen peroxide with chemiluminescent signal formation catalyzed by peroxygenase from the fungus Agrocybe aegerita V.Brig.

Conditions of luminol oxidation by hydrogen peroxide in the presence of peroxygenase from the mushroom Agrocybe aegerita V.Brig. have been optimized. The pH value (8.8) at which fungal peroxygenase produces a maximum chemiluminescent signal has been shown to be similar to the pH optimum value of horseradish peroxidase. Luminescence intensity changed when the concentration of Tris-buffer was varied; maximum intensity of chemiluminescence was observed in 40 mM solution. It has been shown that enhancer (p-iodophenol) addition to the substrate mixture containing A. aegerita peroxygenase exerted almost no influence on the intensity of the chemiluminescent signal, similarly to soybean, palm, and sweet potato peroxidases. Detection limit of the enzyme in the reaction of luminol oxidation by hydrogen peroxide was 0.8 pM. High stability combined with high sensitivity make this enzyme a promising analytical reagent.     

The effect of antioxidants on the formation of free radicals and primary products of the peroxidase reaction

Antioxidants suppress the formation of radicals in peroxidase processes. The chemiluminescence kinetic curves in the horseradish peroxidase/luminol/H₂O₂ system have been compared with those obtained from the mathematical model of the reaction. It was shown that the effect of trolox, ascorbate, and mexidol is a result of the reaction of the luminol radical with the inhibitor molecule (rate constants, 1.0·10¹⁰, 9.0·10⁹, and 2.3·10⁵ mol^–1 min^–1, respectively). The antiradical action of quercetin has been described by eight reactions that were based on the assumption of two reaction centers in the molecule, each reacting with two radicals. The hypothesis that the antioxidant molecule captures the enzyme intermediate radicals in peroxidase cycle, rather than the radical of the reaction product was not confirmed because the calculated curves differed from the experimental point positions. Apparently, the formation of radicals in the presence of peroxidases in living cells and the subsequent events, such as apoptosis, may be prevented not only by the inhibition of an enzyme but also by antioxidants that capture free-radical reaction products     

Improvement of mimetic peroxidase activity of gold nanoclusters on the luminol chemiluminescence reaction by surface modification with ethanediamine

Peroxidase is a commonly used catalyst in luminol–H₂O₂ chemiluminescence (CL) reactions. Natural peroxidase has a sophisticated separation process, short shelf life and unstable activity, therefore it is important to develop peroxidases that have both high catalytic activity and good stability as alternatives to the natural enzyme. Gold nanoclusters (Au NCs) are an alternative peroxidase with catalytic activity in the luminol–H₂O₂ CL reaction. In the present study, ethanediamine was modified on the surface of Au NCs forming cationic Au NCs. The zeta potential of the cationic Au NCs maintained its positive charge when the pH of the solution was between 4 and 9. The cationic Au NCs showed higher catalytic activity in the luminol–H₂O₂ CL reaction than did unmodified Au NCs. A mechanism study showed that the better performance of cationic Au NCs may be attributed to the generation of ¹O₂ on the surface of cationic Au NCs and a positive surface charge, for better affinity to luminol. Cationic Au NC, acting as a peroxidase mimic, has much better stability than horseradish peroxidase over a wide range of temperatures. We believe that cationic Au NCs may be useful as an artificial peroxidase for a wide range of potential applications in CL and bioanalysis.     

Optimization of Peroxidase-Catalyzed Oxidation of Luminol in Reversed Micelles of Surfactants in Octane

Luminol oxidation in the Aerosol OT (AOT) reversed micelles in octane catalyzed by horseradish peroxidase (HRP), or its conjugate with Cortisol (HRP-COR), was optimized. The chemiluminescence intensity during luminol oxidation was strongly dependent on the method of preparation of the reaction mixture and the addition of Triton X-45, cyclohexanol and the chemiluminescence “enhancer”, p-iodophenol, into the micellar system. Five procedures for the preparation of the reaction mixture were compared. The maximum chemiluminescence was observed in the micellar system containing all the reaction components excluding a biocatalyst, addition of which into the system started the reaction. Triton X-45, cyclohexanol or p-iodophenol added to the micellar system enhanced significantly the chemiluminescence intensity. The “enhancing” action of p-iodophenol in AOT reversed micelles was 10-fold less than in an aqueous medium.     

Sensitivity limitations encountered in enhanced horseradish peroxidase catalysed chemiluminescence

Conditions for the enhanced horseradish peroxidase (HRP) catalysed reaction between luminol and hydrogen peroxide were optimized to determine detection limits for HRP conjugated to antibody fragment (HRP-Fab) in solution phase. Light output was linear with respect to HRP-Fab concentration but became nonlinear at low HRP-Fab concentrations when an accelerator (enhancer) of the reaction was used. para-Phenylphenol was a more effective enhancer than p-iodophenol at HRP-Fab concentrations below 20 pmol/l. The detection limit for HRP-Fab was 1.2 femtomoles in the absence of p-phenylphenol and 0.08 femtomole in the presence of p-phenylphenol. The acceleration of peroxidase activity at the lowest HRP-Fab concentrations occurred after an incubation time period of up to five minutes. This lag time limited the sensitivity and the mechanism for it was sought. Preincubation experiment results indicated that the lag time phenomenon may involve a reversible alteration in HRP catalytic activity and that enhancer, peroxide, luminol and HRP-Fab had to be incubated together some time before maximum activation could occur.     

Enhanced chemiluminescence reaction applied to the study of horseradish peroxidase stability in the course of p-iodophenol oxidation

The enhanced chemiluminescence reaction (ECL) was applied to the study of horseradish peroxidase (HRP) inactivation during the oxidation of p-iodophenol. Enzyme inactivation was shown to be the main reason for light decay in the course of the reaction. No individual effect of luminol and p-iodophenol as enhancer on HRP activity towards 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was detected, enzymatic activity loss was detected only in the course of the ECL reaction. HRP activity towards ABTS (a colorimetric substrate) fell in a similar manner to the decay in light emission. The reactive radical species formed during enhancer oxidation were suggested as the main inactivating agents. The similarity of changes in light intensity and enzymatic activity allows one to apply the ECL reaction for testing potential stabilizers of HRP. The loss of enzyme activity can be partially explained by non-specific interaction of radical species with protein globule. The addition of bovine serum albumin provided almost complete protection of peroxidase from inactivation. This confirms the non-specific inactivation with highly reactive endogenous intermediates through the modification of a protein globule. © 1997 John Wiley & Sons, Ltd.     

Flow‐injection chemiluminescence method for the determination of chloramphenicol based on luminol–sodium periodate order‐transform second‐chemiluminescence reaction

A new chemiluminescence (CL) reaction was observed when chloramphenicol solution was injected into the mixture after the end of the reaction of alkaline luminol and sodium periodate or sodium periodate was injected into the reaction mixture of chloramphenicol and alkaline luminol. This reaction is described as an order‐transform second‐chemiluminescence (OTSCL) reaction. The OTSCL method combined with a flow‐injection technique was applied to the determination of chloramphenicol. The optimum conditions for the order‐transform second‐chemiluminescence emission were investigated. A mechanism for OTSCL has been proposed on the basis of the chemiluminescence kinetic characteristics, the UV‐visible spectra and the chemiluminescent spectra. Under optimal experimental conditions, the CL response is proportional to the concentration of chloramphenicol over the range 5.0 × 10^?7–5.0 × 10^?5 mol/L with a correlation coefficient of 0.9969 and a detection limit of 6.0 × 10^?8 mol/L (3σ). The relative standard deviation (RSD) for 11 repeated determinations of 5.0 × 10^?6 mol/L chloramphenicol is 1.7%. The method has been applied to the determination of chloramphenicol in pharmaceutical samples with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

An enhanced chemiluminescent enzyme immunoassay for serum carcinoembryonic antigen based on a modification of a commercial kit

A conventional colorimetric peroxidase end-point (ortho-phenylenediamine substrate), used in an enzyme immunoassay for carcinoembryonic antigen, employing plastic beads as solid support, has been replaced by a much faster (30 seconds versus 30 minutes) enhanced chemiluminescent assay for the peroxidase label. Para-iodophenol was used to enhance the light emission from the peroxidase catalysed chemiluminescent reaction between luminol and hydrogen peroxide. Values for precision and carcinoembryonic antigen concentration obtained with the chemiluminescent and colorimetric versions of the immunoassay on 62 serum specimens were in good agreement.     

DPPH·–luminol chemiluminescence system and its application in the determination of scutellarin in pharmaceutical injections and rat plasma with flow injection analysis

In this article, a DPPH·–luminol chemiluminescence (CL) system was reported and the CL mechanism was discussed according to the CL kinetic properties after sequence injecting DPPH· into the DPPH·–luminol reaction mixture. It was observed that scutellarin could inhibit the CL response of the DPPH·–luminol system. Based on this observation, a simple and rapid flow injection CL method was developed for the determination of scutellarin using the inhibition effect in alkaline medium. The optimized chemical conditions for the CL reaction were 5 × 10^?6 mol/L DPPH · and 1.0 × 10^?4 mol/L luminol in 0.01 mol/L NaOH. Under optimized conditions, the CL intensity was inversely proportional to the concentration of scutellarin over the ranges 5–2000 and 40–3200 ng/ml in pharmaceutical injection and rat plasma, respectively. The limits of detection (S/N  = 3) were 5 and 40 ng/ml in preparations and rat plasma, respectively. Furthermore, the precision, recovery and stability of the validated method were acceptable for the determination of scutellarin in both pharmaceutical injections and rat plasma. The presented method was successfully applied in the determination of scutellarin in pharmaceutical injections and real rat plasma samples.